Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and are the most potent toxins known. These toxins are a well-recognized source of food poisoning, often resulting in serious harm or even death of the victims. There are seven structurally related botulinum neurotoxins or serotypes (BoNT/A-G), each of which is composed of a heavy chain (−100 KD) and a light chain (−50 KD). The heavy chain mediates toxin entry into a target cell through receptor-mediated endocytosis. Once internalized, the light chain is translocated from endosomal vesicle lumen into cytosol, and acts as a zinc-dependent protease to cleave proteins that mediate vesicle-target membrane fusion (“substrate proteins”). Cleavage of SNARE proteins blocks vesicle fusion with plasma membrane and abolishes neurotransmitter release at neuromuscular junction.
These BoNT substrate proteins include plasma membrane protein syntaxin, peripheral membrane protein SNAP-25, and a vesicle membrane protein synaptobrevin (Syb). These proteins are collectively referred to as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Among the SNARE proteins, syntaxin and SNAP-25 usually reside on the target membrane and are thus referred to as t-SNAREs, while synaptobrevin is found exclusively with synaptic vesicles within the synapse and is called v-SNARE. Together, these three proteins form a complex that are thought to be the minimal machinery to mediate the fusion between vesicle membrane and plasma membrane. BoNT/A, E, and C1 cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), at single but different sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.
Botulinum neurotoxins are listed as a bioterror threat due to their extreme potency and the lack of immunity in the population. Because of their paralytic effect, low dose of botulinum neurotoxin has also been used effectively to treat certain muscle dysfunctions and other related diseases in recent years.
Due to their threat as a source of food poisoning, and as bioterrorism weapons, there is a need to sensitively and speedily detect BoNTs. Currently, the most sensitive method to detect toxins is to perform toxicity assay in mice. This method requires large numbers of mice, is time-consuming and cannot be used to study toxin catalytic kinetics. A number of amplified immunoassay systems based on using antibodies against toxins have also been developed, but most of these systems require complicated and expensive amplification process, and cannot be used to study toxin catalytic activity either. Although HPLC and immunoassay can be used to detect cleaved substrate molecules and measure enzymatic activities of these toxins, these methods are generally time-consuming and complicated, some of them require specialized antibodies, making them inapplicable for large scale screening. Therefore, there is a need for new and improved methods and compositions for detecting BoNTs.
There is also a need for improved technique for screening for inhibitors of BoNTs. These inhibitors can be used as antidotes to the toxins for both preventive and treatment purposes.
Recently, a new approach based on intramolecular quenching of fluorigenic amino acid derivatives has been explored. In principle, two amino acid derivatives are used to replace two native amino acids in a very short synthetic peptide (20-35 amino acids) that containing toxin cleavage sites. The fluorescence signal of one amino acid derivative is quenched by another amino acid derivative when they are close to each other in the peptide. Cleavage of the peptide separates two amino acid derivatives and an increase in fluorescence signal can be detected (Schmidt J, Stafford R, Applied and Environmental microbiology, 69:297, 2003). This method has been successfully used to characterize a BoNT/B inhibitor. However, it requires synthesis of peptides with modified amino acid derivatives and is not suitable for use in living cells.